Anthrax lethal factor is a MAPK kinase protease

ABSTRACT

The present invention relates to in vitro and ex vivo methods of screening for modulators, homologues, and mimetics of lethal factor mitogen activated protein kinase kinase (MAPKK) protease activity, as well as methods of treating cancer by administering LF to transformed cells.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. S No. 60/080,330, filedApr. 1, 1998, herein incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] The present invention relates to in vitro and ex vivo methods ofscreening for modulators, homologues, and mimetics of lethal factormitogen activated protein kinase kinase (MAPKK) protease activity, aswell as methods of treating cancer by administering LF to tranformedcells.

BACKGROUND OF THE INVENTION

[0004] Anthrax toxin, produced by Bacillus anthracis, is composed ofthree proteins: protective antigen (PA), edema factor (EF), and lethalfactor (LF) (Leppla, Handbook of Natural Toxins 8:543-572 (Moss et al.,eds., 1995)). PA alone has no toxic effect upon cells, but instead bindsto specific cell surface receptors. Upon proteolytic activation to a63-kDa fragment (PA63), PA forms a heptameric membrane-inserted channel,which mediates the entry of EF and LF into the cytosol via the endosomalpathway (Gordon et al., Infect. Immun. 56:1066-1069 (1988); Milne etal., J. Biol Chem. 269:20607-20612 (1994)). Thus, EF or LF are toxic tocells when combined with PA.

[0005] EF is an adenylate cyclase, and together with PA forms a toxinreferred to as edema toxin (Leppla, Proc. Natl. Acad. Sci. USA79:3162-3166 (1982)). LF and PA together form a toxin referred to aslethal toxin (“LT”). Until the present discovery, however, the specificactivity of LF in the cell was unknown. Lethal toxin is the dominantvirulence factor produced by B. anthracis and is the major cause ofdeath of infected animals (Pezard et al., Infect. Immun. 59:3472-3477(1991)). Intravenous injection of lethal toxin causes death of Fisher344 rats in as little as 38 minutes (Ezzell et al., Infect Immun.45:761-767 (1984)), and incubation in vitro with mouse macrophagescauses lysis in 90-120 minutes (Friediander, J. Biol Chem. 261:7123-7126(1986)).

[0006] LF contains a limited sequence homology to a putativezinc-binding site at residues 686-690, HEFGH, characteristic ofmetalloproteases (Klimpel et al., Mol. Microbiol. 13:1093-1100 (1994)).Substitution of the H or E residues inactivates LF (e.g., as in therecombinant LF mutant E687C) (Klimpel et al., 1994, supra) and decreasesits binding of zinc (Klimpel et a!., 1994, supra; Kocki et al., FEMSMicrobiol. Lett. 124:343-348 (1994)). Certain metalloprotease inhibitorsalso protect macrophages against lethal toxin (Klimpel et al., 1994,supra; Menard et al., Biochem J. 320:687-691 (1996)). However, nophysiological substrate has been identified for LF, and LF proteaseactivity has not been demonstrated.

SUMMARY OF THE INVENTION

[0007] The present invention thus identifies anthrax lethal factor (LF)as a protease, which acts as an inhibitor of the mitogen activatedprotein kinase (MAPK) signal transduction pathway. The present inventionalso identifies specific substrates for LF protease activity. Forexample, LF cleaves MAPK kinases 1, 2, and 3 (MEK) at specific sites intheir N-termini, thereby preventing activation of MAPK (ERK2). LF isthus useful for inhibition of cancer cells that have an activated MAPKsignal transduction pathway. Furthermore, the present invention providesmeans for assaying in vivo and in vitro for modulators and mimetics ofLF, for use in treating cancer.

[0008] In one aspect, the present invention provides an in vitro methodfor screening modulators of lethal factor (LF) mitogen activated proteinkinase kinase (MAPKK) protease activity, the method comprising the stepsof: (i) providing LF in an aqueous solution, wherein the LF has MAPKKprotease activity in the solution; (ii) contacting LF with substancessuspected of having the ability to modulate MAPKK protease activity; and(iii) assaying for the level of LF MAPKK protease activity.

[0009] In another aspect, the present invention provides a kit forscreening in vitro for modulators of lethal factor (LF) mitogenactivated protein kinase kinase (MAPKK) protease activity, the kitcomprising; (i) a container holding LF, wherein the LF has MAPKKprotease activity; and (ii) instructions for assaying for LF MAPKKprotease activity.

[0010] In another aspect, the present invention provides an in vivomethod for screening modulators of lethal factor (LF) mitogen activatedprotein kinase kinase (MAPKK) protease activity, the method comprisingthe steps of: (i) contacting a living cell with LF, wherein the LF hasMAPKK protease activity; (ii) contacting the cell with substancessuspected of having the ability to modulate MAPKK protease activity; and(iii) assaying for the level of LF MAPKK protease activity.

[0011] In another aspect, the present invention provides an in vitromethod for screening mimetics of lethal factor (LF) having mitogenactivated protein kinase kinase (MAPKK) protease activity, the methodcomprising the steps of: (i) providing a compound suspected of being anLF mimetic in an aqueous solution; and (ii) assaying for the level ofMAPKK protease activity.

[0012] In another aspect, the present invention provides an in vivomethod for screening for mimetics of lethal factor (LF) having mitogenactivated protein kinase kinase (MAPKK) protease activity, the methodcomprising the steps of: (i) contacting a living cell with a compoundsuspected of being an LF mimetic; and (ii) assaying for the level ofMAPKK protease activity.

[0013] In another aspect, the present invention provides a method forinhibiting proliferation of a cancer cell, the method comprising thestep of contacting the cell with LF, wherein the LF has MAPKK proteaseactivity.

[0014] In one embodiment, the LF is recombinant. In another embodiment,the MAPKK1 or MAPKK2 is recombinant. In another embodiment, therecombinant MAPKK1 or recombinant MAPKK2 is linked to a detectablemoiety.

[0015] In one embodiment, the assay is a Mos-induced activation of MAPKassay in a Xenopus oocyte. In another embodiment, the assay is an MAPKK1or MAPKK2 mobility assay. In another embodiment, the assay is an MBPphosphorylation assay.

[0016] In one embodiment, the step of contacting the cell comprisingtransducing the cell with an expression vector encoding LF. In anotherembodiment, the step of contacting further comprises contacting a cellwith LF in the presence of protective antigen (PA). In anotherembodiment, the PA is a fusion protein targeted to the cancer cell.

[0017] In another embodiment, the mitogen activated protein kinase(MAPK) signal transduction pathway is activated in the cell.

[0018] In one embodiment, the cell is a human cell. In anotherembodiment, the cell is a Xenopus oocyte. In another embodiment, thecell is a cancer cell. In another embodiment, the cancer cell is from asarcoma. In another embodiment, the cell is from a transformed cellline. In another embodiment, the cell line is transformed with Ras.

[0019] In another aspect, the invention provides methods for reversing atransformed phenotype in a cell by treating the cell with LT. In oneembodiment, morphological changes associated with transformation arereversed. In another embodiment, the diffuse pattern of actindistribution that is characteristic of transformed cells is reversed. Inanother embodiment, the rate and extent of proliferation of atransformed cell is inhibited. In another embodiment, the ability of atransformed cell to grow independently of anchorage to a substrate isreversed.

[0020] In another aspect, the invention provides a method foridentifying a three-dimensional structure of MAPKK or LF proteins, themethod comprising the steps of: (i) receiving input of at least 10contiguous amino acids of the amino acid sequence of MAPKK or LF, or atleast 30 contiguous nucleotides of the nucleotide sequence of a geneencoding MAPKK or LF, and conservatively modified variants thereof; and(ii) generating a three-dimensional structure of the protein encoded bythe amino acid sequence.

[0021] In one embodiment, the amino acid sequence is a primary structureand the generating step includes the steps of: (i) forming a secondarystructure from said primary structure using energy terms encoded by theprimary structure; and (ii) forming a tertiary structure from saidsecondary structure using energy terms encoded by said secondarystructure. In another embodiment, the generating step includes the stepof forming a quaternary structure from said tertiary structure usinganisotropic terms encoded by the tertiary structure. Another embodimentfurther comprises the step of identifying regions of thethree-dimensional structure of the protein that bind to ligands andusing the regions to identify ligands that bind to the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1: Alignment of the N-terminal amino acids of MAPKK 14. TheN-terminal 60 amino acids of Xenopus (X) MAPKK1, mouse (M) MAPKK1, aswell as human (H) MAPKK 14 were aligned using the Multiple SequenceAlignment tool of the Institute for Biomedical Computing, WashingtonUniversity, St. Louis, accessible through the internet(http://www.ibc.wustl.edu/ibc/msa.html).

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0023] I. Introduction

[0024] The present invention identifies anthrax lethal factor (LF) as aninhibitor of the mitogen activated protein kinase (MAPK) signaltransduction pathway. LF specifically cleaves MAPKK, which is a kinasecomponent of the MAPK cascade. The mitogen activated protein kinase(MAPK) signal transduction pathway is involved in cell proliferation anddifferentiation. This pathway also plays a crucial role in regulatingoocyte meiotic maturation (Moriguchi et al., Adv. Pharmacol. 36:121-137(1996); Murakami et al., Methods in Enzymology 283:584-600 (Dunphy, ed.,1997); Matten et al., Seminars in Dev. Biol. 5:173-181 (1994)).

[0025] Constitutive activation of signal transduction pathways can leadto cell transformation. The MAPK signal transduction pathway involves acascade of kinases, in which MAPKK phosphorylates and activates MAPK,and MAPK phosphorylates and activates pp90^(rsk) (see, e.g., Stugill etal., Nature 334:715 (1988); Gomez & Cohen, Nature 353:170 (1991)). Inaddition, cellular forms of oncogenes participate in this pathway assignalling components upstream of the MAPK phosphorylation cascade,e.g., Ras, Raf, and Mos. Furthermore, a variety of cytokines thatinteract with cell surface receptors have the ability to activate thispathway, e.g., IL1 and TNF. As described herein, LF and its homologues,modulators, and mimetics are thus useful for inhibiting theproliferation of cancer cells.

[0026] LF inhibits the MAPK pathway via protease activity, by cleavingits substrate, mitogen activated protein kinase kinase (MAPKK). Thus,the discovery that LF is a protease that inhibits the MAPK signaltransduction pathway provides means for identifying novel therapeuticagents such as LF mimetics that inhibit the MAPK signal transductionpathway. Such agents are useful for treating cancer. Indeed, it has beendiscovered that LF and PA can reverse a transformed phenotype in cells.As shown in Example VII, infra, LF and PA can reverse numerous cellularproperties associated with transformation, including, but not limitedto, morphological features, intracellular patterns of actindistribution, proliferation rates, and anchorage-independent growth.

[0027] In addition, LF and LF mimetics that are modified to specificallytarget cancer cells are particularly useful as cancer therapeutics. Thisinvention also provides means for specifically assaying for modulatorsof LF activity, e.g., inhibitors and activators. For example, LFinhibitors are useful as therapeutic agents for B. anthracis infectionand inhibitors of anthrax lethal toxin. LF activators may be useful toenhance LF or LF mimetic activity for treatment of cancer. Such mimeticsand modulators of LF can be identified using high throughput assaytechniques, using the assays described herein.

[0028] II. Definitions

[0029] Unless defined otherwise, all technical and scientific terms usedherein have the meaning commonly understood by one of ordinary skill inthe art to which this invention belongs.

[0030] The “mitogen activated protein kinase (MAPK) pathway” is a signaltransduction pathway that effects gene regulation, which controls cellproliferation and differentiation in response to extracellular signals.This pathway also involved in oocyte meiotic maturation. The MAPKpathway is found, e.g., in frogs, and in mammals, e.g., mice, rats, andhumans. This pathway can be “activated” by cytokines such as I1-1 andTNF, and constitutively activated by proteins such as Mos, Raf, Ras, andV12HaRas, (see, e.g., Moriguchi et al., Adv. Pharmacol. 36:121-137(1996); Murakami & Vande Woude, in Methods in Enzymology 283:584-600(Dunphy, ed., 1997); Matten & Vande Woude, Seminars in DevelopmentalBiol. 5:173-181 (1994); White et al., Cell 80:533-541 (1995);Ruckdeschel et al., J. Biol. Chem. 272:15920-15927 (1997); West et al.,J. Leukoc. Biol. 91:88-95 (1997); Winston et al., J. Immunol.155:1525-1533 (1995); Hambleton et al., J. Exp. Med. 182:147-154 (1995);Ridley et al., J. Immunol. 158:3165-3173 (1997); Lu et al., Neurochem.Int. 30:401-410 (1997); Guan et al., J. Biol. Chem. 272:8083-8089(1997); Scherle et al., Biochem. Biophys. Res. Commun. 230:573-577(1997); Huwiler et al., FEBS Lett. 350:135-138 (1994); and Bird et al.,FEBS Lett. 338:31-36 (1994)).

[0031] “Mitogen activated protein kinase kinase (MAPKK)” refers to afamily of protein kinases that are part of the mitogen activated proteinkinase (MAPK, also known as ERK) signal transduction pathway, e.g.,MAPKK1, MAPKK2, MAPKK3 (also known as MEK). These proteins sharesequence similarity and are cleaved near the N-terminus by LF (see FIG.1). The term MAPKK thus refers to members of the MAPKK family, e.g.,MAPKK1, MAPKK2, and MAPKK3, and conservatively modified variantsthereof. The term also includes polymorphic variants, alleles, mutantsand interspecies homologues with greater than about 60% sequencehomology to MAPKKs 1-3 (see discussion of MAPKK Genebank deposit,below).

[0032] “Mitogen activated protein kinase kinase protease activity”(MAPKK, also known as MEK) or “LF MAPKK protease activity” refers theactivity of a molecule, e.g., LF, an LF homologue, or an LF mimetic thathas the ability to specifically cleave members of the MAPKK family,e.g., MAPKK1, MAPKK2, MAPKK3, at the N-terminus.

[0033] Anthrax “lethal factor” or “LF” is a protein that is naturallyproduced by B. anthracis and that has MAPKK protease activity. As usedherein, the term LF includes naturally occurring LF, recombinant LF, andfunctional LF equivalents that have MAPKK protease activity. The term LFtherefore refers to LF homologues such as polymorphic variants, alleles,mutants, and closely related interspecies variants that have about atleast 60% amino acid sequence identity to LF (e.g., are substantiallyidentical to LF; see Genebank sequence deposit, below) and have MAPKKprotease activity, as determined using the assays described herein.Deletion analysis of LF shows that the PA binding domain is at theamino-terminus of LF, and that amino-terminal residues 1-254 of LF aresufficient for PA binding activity (Arora et al., J. Biol. Chem.268:3334-3341 (1993)). When LF is administered with PA, LF preferablyincludes the PA binding domain.

[0034] An “LF mimetic” refers to a compound or molecule, e.g., apeptide, polypeptide, or small chemical molecule, that recognizes MAPKKas a substrate and cleaves MAPKK at the same site as LF. LF mimeticsthus include LF homologues. LF mimetics would also include small LFpeptides that retained the LF MAPKK protease active site, andconservatively modified variants thereof, as well as truncated versionsof LF that retained LF MAPKK activity. Small chemical molecules thatmimic the LF active site are also LF mimetics. LF mimetics are testedusing assays for LP activity, e.g., MAPKK mobility assays, MOS-inducedactivation of MAPK in oocytes and myelin basic protein (MBP)phosphorylation, as described below. When testing for an LF mimetic, LFis typically used as a positive control for MAPKK protease activity. Arelative activity value is assigned to LF, e.g., 100. Mimic activity isachieved when mimetic MAPKK protease activity relative to the control isabout 25, more preferably 50-100.

[0035] One example of a potential LF mimetic is the compound PDO9859,[2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one], identified as aninhibitor of the MAPK pathway (Dudley et al., Proc. Nat'l Acad. Sci. USA92:7686 (1995)). A screen of the National Cancer Institute's 60 cellline in vitro anti-neoplastic drug database (Weinstein et al., Science275:343 (1997); Koo et al., Cancer Res. 56:5211 (1996)) revealed thatthis compound has an activity profile that is similar to LF.

[0036] The anthrax “protective antigen” (PA) is protein produced byBacillus anthracis. PA is one of two protein components of the lethal oranthrax toxin produced by B. anthracis. The 83 kDa PA binds at itscarboxyl-terminus to a cell surface receptor, where it is specificallycleaved by a protease, e.g., furin, clostripain, or trypsin. Thisenzymatic cleavage releases a 20 kDa amino-terminal PA fragment, while a63 kDa carboxyl-terminal PA fragment remains bound to the cell surfacereceptor. The 63 kDa fragment is also referred to as “processedprotective antigen.” Processed PA contains both a cell surface receptorbinding site at its carboxyl-terminus and a lethal factor binding siteat its new amino-terminus (see, e.g., Singh et al., J. Biol. Chem.264:19103-19107 (1989)). Processed PA may be produced by enzymaticcleavage in vitro, ex vivo, or in vivo, or as a recombinant protein. Asused herein the term PA refers PA molecules that have the lethal factorbinding site, e.g., recombinant PA, naturally occurring PA, functionalequivalents of PA that contain the lethal factor binding site, and PAfusion proteins that contain the lethal factor binding site.

[0037] As described in U.S. Pat. Nos. 5,591,631 and 5,677,274 (hereinincorporated by reference in their entirety), PA fusion proteins can bemade that target PA to particular cells, such as cancer cells and HIVinfected cells, using PA fusion proteins comprising ligands forreceptors that are specifically expressed on the target cell.

[0038] “Lethal toxin”, or “LT”, refers to the combination of PA and LF.

[0039] “Modulators of LF MAPKK protease activity” refers to activatingor inhibitory molecules identified using in vitro and in vivo assays forLF MAPKK activity. Such assays include, e.g., MAPKK mobility assays,MOS-induced activation of MAPK in oocytes, myelin basic protein (MBP)phosphorylation, morphological changes, immortalization of cells,aberrant growth control, anchorage dependence, proliferation,malignancy, contact inhibition and density limitation of growth, growthfactor or serum dependence, tumor specific markers levels, invasiveness,tumor growth, and the like, in vitro, in vivo, and ex vivo as describedbelow. Potential modulators include peptides, polypeptides, and smallchemical molecules. The ability of LF, its homologues, mimetics, andmodulators to inhibit proliferation of cancer cells can also bedetermined using the assays described herein.

[0040] Samples or assays that are treated with a potential LF MAPKKprotease modulators or LF compounds that are used to treat cancer arecompared to control samples without the test compound, to examine theextent of inhibition or activation of LF MAPKK protease activity.Control samples (untreated with test inhibitors or activators) areassigned a relative LF MAPKK protease activity value of 100. Inhibitionof LF MAPKK protease activity is achieved when the LF MAPKK proteaseactivity value relative to the control is about 75, preferably 50, morepreferably 25. Activation is achieved when the LF MAPKK proteaseactivity value relative to the control is about 150, more preferably200.

[0041] A “Mos-induced activation assay” refers to an assay for LF MAPKKprotease activity that tests for inhibition of oocyte maturation aftertreatment with LF, typically by examining inhibition of germinal vesiclebreakdown (GVBD).

[0042] A “MAPKK mobility assay” refers to an assay for LF MAPKK proteaseactivity that tests for changes in MAPKK electrophoretic mobility aftertreatment with LF.

[0043] A “myelin basic protein (MBP) phosphorylation assay” refers to anassay for LF MAPKK protease activity that tests for inhibition of myelinbasic protein (MBP) phosphorylation after treatment with LF.

[0044] The phrase “contacting a cell” refers to any method whereby LF,an LF homologue, modulator, or an LF mimetic is introduced into a cell,e.g., by transduction of a nucleic acid encoding LF or an LF homologueor mimetic, by administering LF in the presence of PA to the cellmedium, by injecting LF or an LF homologue or mimetic into the cell, byconjugating LF or an LF homologue or mimetic to a molecule, e.g., areceptor ligand, that allows LF or the LF homologue or mimetic to betranslocated into a cell, and by introducing LF or the LF homologue ormimetic into a cell using a vehicle such as a liposome.

[0045] “Transduction” refers to any method whereby a nucleic acid isintroduced into a cell, e.g., by transfection, lipofection,electroporation, biolistics, passive uptake, lipid:nucleic acidcomplexes, viral vector transduction, injection, naked DNA, and thelike.

[0046] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0047] “In vivo” refers to assays that are performed using living cells,including cells in an animal and cells ex vivo.

[0048] “Ex vivo” refers to assays that are performed using a cell withan intact membrane that is outside of the body, e.g., explants, culturedcell lines, transformed cell lines, primary cell lines, and extractedtissue, e.g., blood, oocytes.

[0049] “In vitro” refers to assays that do not require the presence of acell with an intact membrane.

[0050] A “cancer cell” refers to a cancerous, pre-cancerous ortransformed cell, either in vivo, ex vivo, and in tissue culture, thathas spontaneous or induced phenotypic changes that do not necessarilyinvolve the uptake of new genetic material. Although transformation canarise from infection with a transforming virus and incorporation of newgenomic nucleic acid, or uptake of exogenous nucleic acid, it can alsoarise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation/cancer is associated with,e.g., morphological changes, immortalization of cells, aberrant growthcontrol, foci formation, anchorage dependence, proliferation,malignancy, contact inhibition and density limitation of growth, growthfactor or serum dependence, tumor specific markers levels, invasiveness,tumor growth or suppression in suitable animal hosts such as nude mice,and the like, in vitro, in vivo, and ex vivo (see Example VII) (see alsoFreshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed.1994)).

[0051] A “sarcoma” refers to a type of cancer cell that is derived fromconnective tissue, e.g., bone (osteosarcoma) cartilage (chondrosarcoma),muscle (rhabdomyosarcoma or rhabdosarcoma), fat cells (liposarcoma),lymphoid tissue (lymphosarcoma), collagen-producing fibroblasts(fibrosarcoma). Sarcomas may be induced by infection with certainviruses, e.g., Kaposi's sarcoma, Rous sarcoma virus, etc.

[0052] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogues ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analoguesinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0053] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

[0054] The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analogue or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.

[0055] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogues and amino acid mimeticsthat function in a manner similar to the naturally occurring aminoacids. Naturally occurring amino acids are those encoded by the geneticcode, as well as those amino acids that are later modified, e.g.,hydroxyproline, alpha-carboxyglutamate, and O-phosphoserine. Amino acidanalogues refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an alpha carbonthat is bound to a hydrogen, a carboxyl group, an amino group, and an Rgroup (e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium). Such analogues have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. Amino acidmimetics refer to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunction in a manner similar to a naturally occurring amino acid.

[0056] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0057] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences, orwhere the nucleic acid does not encode an amino acid sequence, toessentially identical sequences. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because ofthe degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

[0058] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologues, and alleles of the invention.

[0059] The following groups each contain amino acids that areconservative substitutions for one another:

[0060] 1) Alanine (A), Glycine (G);

[0061] 2) Serine (S), Threonine (T);

[0062] 3) Aspartic acid (D), Glutamic acid (E);

[0063] 4) Asparagine (N), Glutamine (Q);

[0064] 5) Cysteine (C), Methionine (M);

[0065] 6) Arginine (R), Lysine (K), Histidine (H);

[0066] 7) Isoleucine (1), Leucine (L), Valine (V); and

[0067] 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0068] (see, e.g., Creighton, Proteins (1984)).

[0069] A “detectable moiety” or label” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available.

[0070] A protein that is “linked to a detectable moiety” is one that isbound, either covalently, through a linker, or through ionic, van derWaals or hydrogen bonds to a label such that the presence of the proteinmay be detected by detecting the presence of the label or detectablemoiety bound to the protein.

[0071] The term “recombinant” when used with reference, e.g., to a cell,nucleic acid, vector, or protein indicates that the cell, nucleic acid,or vector has been modified by the introduction of a heterologousnucleic acid or the alteration of a native nucleic acid, or that thecell is derived from a cell so modified, or that the protein is encodedor expressed by such a nucleic acid or cell. Thus, for example,recombinant cells express genes and proteins that are not found withinthe native (non-recombinant) form of the cell or express native genesand proteins that are otherwise abnormally expressed, under expressed ornot expressed at all.

[0072] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides (i.e., 60% identity)that are the same, when compared and aligned for maximum correspondenceover a comparison window, as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Preferably, the percent identity exists over a region of thesequence that is at least about 25 amino acids in length, morepreferably over a region that is 50 or 100 amino acids in length.

[0073] For sequence comparison, one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

[0074] A “comparison window,” as used herein, includes reference to asegment of contiguous positions selected from the group consisting offrom 20 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection.

[0075] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nucleic Acids Res. 12:387-395 (1984)).

[0076] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N—4, and a comparison of both strands.

[0077] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0078] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid. Thus,a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two molecules or theircomplements hybridize to each other under stringent conditions, or canbe amplified by the same primer set.

[0079] III. In vitro, in vivo, and ex vivo Assays for Modulators,Homologues, and Mimetics of LF Activity

[0080] The present invention provides assays to identify modulators ofLF MAPKK protease activity and LF mimetics of this activity. The presentinvention also provides assays to determine suitable compounds for usein cancer therapeutics, e.g., LF, LF homologues and mimetics, and LFmodulators. A variety of assays can be used to test the compounds of theinvention; these assays examine phosphorylation, MAPKK cleavage, oroocyte maturation, e.g., ex vivo and in vitro Mos-activation of the MAPKpathway in oocytes (Example I); in vitro and ex vivo phosphorylation ofmyelin basic protein (MBP) or MAPK (Examples I and II);, ex vivo and invitro cleavage of MAPKK and determination of electrophoretic mobility(Examples II, IV, and V), ex vivo alteration of phenotypiccharacteristics of transformed cells (Example VII); and other phenotypicchanges in transformed or cancer cells, such as the ability to grow onsoft agar; changes in contact inhibition and density limitation ofgrowth; changes in growth factor or serum dependence; changes in thelevel of tumor specific markers; changes in invasiveness into Matrigel;changes in tumor growth in vivo, such as in transgenic mice, etc.

[0081] As a general assay format, samples that are with treated LF or LFhomologues or mimetics and with potential LF inhibitors or activatorsare compared to control samples without the test compound, to examinethe extent of modulation. Control samples (untreated with activators orinhibitors) are assigned a relative LF activity value of 100. Inhibitionof LF is achieved when the LF activity value relative to the control isabout 75, preferably 50, more preferably 25. Activation of LF isachieved when the LF activity value relative to the control is about150, preferably 200 or higher. Activation is achieved when the LF MAPKKprotease activity value relative to the control is about 150, morepreferably 200. Inactive mutants of LF, e.g., LF E687C can be used asnegative controls for LF activity, while LF is typically used as apositive control.

[0082] As a general assay format for LF mimetics and homologues,potential mimetics and homologues are tested using assays for LFactivity, e.g., MAPKK mobility or cleavage assays, Mos-inducedactivation of the MAPK pathway in oocytes and myelin basic protein (MBP)or MAPK phosphorylation, as described below. When testing for an LFmimetic or homologue, LF is typically used as a positive control forMAPKK protease activity. A relative activity value is assigned to LF,e.g., 100. Mimic activity is achieved when mimetic or homologue MAPKKprotease activity relative to the control is about at least 25, morepreferably 50-100, or above 100, e.g., 500.

[0083] A. In vivo and ex vivo Assays for Modulators, Homologues, andMimetics of LF

[0084] In vivo and ex vivo assays can be used to identify modulators ofLF MAPKK protease activity or LF mimetics and homologues. The assays ofthe invention used, e.g., oocytes, in which Mos activation of the MAPKpathway is examined, or transformed cells that have activated MAPKpathways. These transformed cells typically express naturally occurringMAPKK, although they may also express recombinant MAPKK. The transformedcells or oocytes may be contacted with naturally occurring LF,recombinant LF, or LF homologues or mimetics, or the cells can expressrecombinant LF or recombinant LF homologues and mimetics, as describedbelow. As described below, modulators, LF homologues, and LF mimeticsare peptides, proteins, and small chemical molecules.

[0085] MAPKK, LF, and LF homologues and mimetics can be administered toa transformed cell or oocyte, e.g., by transduction with an expressionvector encoding MAPKK, LF or an LF homologue or mimetic; by injection ofMAPKK, LF or an LF homologue or mimetic; by administering MAPKK, LF oran LF homologue or mimetic in a liposome; by creating a targeted fusionprotein with MAPKK, LF or an LF homologue or mimetic; and by contactinga cell, in the presence of PA, with LF or an LF homologue or mimeticthat has the ability to bind to PA. When a cell is contacted with LF inthe presence of PA, the PA binds to its cellular receptor, binds to LF,then internalizes LF into the cell. PA fusion proteins can also be usedto introduce LF in cells. Such PA fusion proteins need only retain theLF binding site on PA, while the portion of PA that binds to itscellular receptor can be replaced by another cellular ligand. The fusionproteins can be targeted to a variety of cellular receptors (see, e.g.,U.S. Pat. Nos. 5,677,274 and 5,591,631). In particular, such fusionproteins are useful for targeting LF to cancer cells, for inhibition ofthe MAPK pathway.

[0086] When assaying for LF modulators and LF mimetics, the testcompounds are added in test concentrations to the cell or oocyte, asdescribed above. For example, modulators can be added to cell media inaqueous solutions or organic solvents such as DMSO, for cellular uptake.Modulators and mimetics can also be administered by injection, by fusionproteins, by liposome delivery, by viral transduction, by transfection,by expression vectors, etc.

[0087] In oocytes, synthesis of Mos activates the MAPK pathway. Insulinalso activates the MAPK pathway. This pathway is essential foractivation of maturation promoting factor (MPF) and the resumption ofmeiosis, i.e., maturation. Oocytes can be isolated from any convenientsource according to standard methods, e.g., frog, fish, or mammalian,e.g., mouse or bovine oocytes. After LF or an LF homologue or mimetic isintroduced to the oocytes, optionally with a modulator, the oocytes areinduced to mature, e.g., for Xenopus oocytes, with progesterone orinsulin, for fish oocytes, with dihydroxyprogesterone. Mammlian oocytesdo not need induction as they spontaneously mature upon isolation.Alternatively, recombinant or naturally occurring Mos can be injectedinto the oocyte to activate the MAPK pathway. The oocytes are thencultured according to standard conditions (see, e.g., Duesbery et al.,Proc. Natl. Acad. Sci. USA 94:9165-9170 (1997); see also Freshney,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)).

[0088] LF modulators, homologues, and mimetics can be assayed byexamining inhibition of germinal vesicle breakdown (GVBD) (see, e.g.,Example I). An intact germinal vesicle signals that maturation, and theMAPK pathway, has been inhibited. Inhibition of GVBD is determined byvisual inspection of the oocytes (using a microscope) for the presenceof the germinal vesicle. The germinal vesicle is seen as a white spot onone side of the oocyte. Inhibition of GVBD can be confirmed by fixingoocytes and manually dissecting to examine whether the germinal vesicleis intact.

[0089] Alternatively, LF modulators, homologues, and mimetics can beidentified by phosphorylation of MAPKK substrates, such MAPK. In suchassays, oocytes are lysed, and the oocyte lysates are subjected toelectrophoresis, and western blots are probed with specific antibodiesagainst phosphorylated MAPK. The oocyte lysates can also be examinedusing ELISA techniques. Optionally, cells can be probed with specificantibodies using in situ techniques.

[0090] In addition, cleavage of MAPKK by LF can be directly detected,using specific antibodies to the truncated MAPKK or by examiningincreased electrophoretic mobility with antibodies to the MAPKKC-terminus. In such assays, oocytes are lysed, and the oocyte lysatesare subjected to electrophoresis, and western blots are probed with asuitable antibody to detect truncated MAPKK. Alternatively, the oocytelysates are examined using ELISA techniques. Oocytes can also be labeledin situ with antibodies that recognize truncated MAPKK.

[0091] Transformed cell lines can also be used ex vivo to identify LFmodulators, homologues, and mimetics (see, e.g., Example II and ExampleVII). The MAPK pathway in the transformed cells can be activated bytreatment with cytokines such as IL1 and TNF-α, as well as othercytokines known to those of skill in the art. Alternatively, cell linesthat have a constitutively activated MAPK pathway can be used. Forexample, such cells include cell lines in which the MAPK pathway isactivated as a result of upstream signalling by oncogenes such as Met,Ras, Raf and Mos, e.g., NIH 3T3 (490) cells, which express the V12HaRasoncogene, IHKE cells transformed with Ras, and NIH3T3 cells transformedwith activated Met. Tumor cell lines or tumor explants with activatedMAPK pathways can also be used in the assays of the invention, e.g.,carcinomas and sarcomas such as osteosarcomas, chondrosarcomas,rhabdomyosarcomas, liposarcomas, lymphosarcomas, and fibrosarcomas.Sarcomas may be induced by infection with certain viruses, e.g.,Kaposi's sarcoma, Rous sarcoma virus, etc.

[0092] LF modulator, homologue, or LF mimetic activity can be examinedthese ex vivo assays by direct detection of MAPKK cleavage. MAPKKcleavage or inhibition of MAPKK cleavage can be detected by any suitablemeans, as described above. For example, cells can be labeled in situwith antibodies that specifically recognize cleaved MAPKK.Alternatively, the cells are lysed and the cellular protein is examinedusing a number of assays, e.g., ELISAs with antibodies specific fortruncated MAPKK or western blots with C-terminal MAPKK antibodies, todetect MAPKK with altered electrophoretic mobility. Cleavage of MAPKKcan also be indirectly monitored by examining phosphorylation of MAPKKsubstrates such as MAPK, as described above, using specific antibodies.

[0093] The following are additional assays that can be used to identifycompounds such as LF, LF homologues, LF mimetics, and LF modulators,which are capable of regulating cell proliferation and tumorsuppression. The phrase “LF constructs” can refer to any of LF and itsalleles, interspecies homologues, polymorphic variants and mutants, aswell as LF mimetics, as used herein. Functional LF homologues, mimetics,and modulators identified by the following assays can then be used ingene therapy to inhibit abnormal cellular proliferation andtransformation.

Soft Agar Growth or Colony Formation in Suspension

[0094] Normal cells require a solid substrate to attach and grow. Whenthe cells are transformed, they lose this phenotype and grow detachedfrom the substrate. For example, transformed cells can grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft agar. The transformed cells, when transfected with LF,regenerate normal phenotype and require a solid substrate to attach andgrow.

[0095] Soft agar growth or colony formation in suspension assays can beused to identify LF homologues, mimetics, and modulators, which whenexpressed in host cells, inhibit abnormal cellular proliferation andtransformation. Typically, transformed host cells (e.g., cells that growon soft agar) are used in this assay. Expression of LF in thesetransformed host cells would reduce or eliminate the host cells' abilityto grow in stirred suspension culture or suspended in semi-solid media,such as semi-solid or soft. This is because the host cells wouldregenerate anchorage dependence of normal cells, and therefore require asolid substrate to grow. Therefore, this assay can be used to identifyLF constructs which reverse the transformed cell phenotype. Onceidentified, such LF constructs and compounds can be used in gene therapyto inhibit abnormal cellular proliferation and transformation.

[0096] Techniques for soft agar growth or colony formation in suspensionassays are described in Freshney, Culture of Animal Cells a Manual ofBasic Technique, 3^(rd) ed., Wiley-Liss, New York (1994), hereinincorporated by reference. See also, the methods section of Garkavtsevet al. (1996), supra, herein incorporated by reference.

Contact Inhibition and Density Limitation of Growth

[0097] Normal cells typically grow in a flat and organized pattern in apetri dish until they touch other cells. When the cells touch oneanother, they are contact inhibited and stop growing. When cells aretransformed, however, the cells are not contact inhibited and continueto grow to high densities in disorganized foci. Thus, the transformedcells grow to a higher saturation density than normal cells. This can bedetected morphologically by the formation of a disoriented monolayer ofcells or rounded cells in foci within the regular pattern of normalsurrounding cells. Alternatively, labeling index with [³H]-thymidine atsaturation density can be used to measure density limitation of growth.See Freshney (1994), supra. The transformed cells, when transfected withLF, regenerate a normal phenotype and become contact inhibited and wouldgrow to a lower density.

[0098] Contact inhibition and density limitation of growth assays can beused to identify LF constructs which are capable of inhibiting abnormalproliferation and transformation in host cells. Typically, transformedhost cells (e.g., cells that are not contact inhibited) are used in thisassay. Expression of an LF construct in these transformed host cellswould result in cells which are contact inhibited and grow to a lowersaturation density than the transformed cells. Therefore, this assay canbe used to identify LF constructs which function as cancer therapeutics.Once identified, such LF constructs can be used in gene therapy toinhibit abnormal cellular proliferation and transformation.

[0099] In this assay, labeling index with [³H]-thymidine at saturationdensity is a preferred method of measuring density limitation of growth.Transformed host cells are transfected with an LF construct and aregrown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with [³H]-thymidine isdetermined autoradiogrpahically. See, Freshney (1994), supra. The hostcells expressing a functional LF construct would give arise to a lowerlabeling index compared to control (e.g., transformed host cellstransfected with a vector lacking an insert).

Growth Factor or Serum Dependence

[0100] Growth factor or serum dependence can be used as an assay toidentify functional LF constructs. Transformed cells have a lower serumdependence than their normal counterparts (see, e.g., Temin, J. Natl.Cancer Insti. 37:167-175 (1966); Eagle et al., J. Exp. Med. 131:836-879(1970)); Freshney, supra. This is in part due to release of variousgrowth factors by the transformed cells. When an LF gene is transfectedand expressed in these transformed cells, the cells would reacquireserum dependence and would release growth factors at a lower level.Therefore, this assay can be used to identify LF constructs whichfunction as cancer therapeutics. Growth factor or serum dependence oftransformed host cells which are transfected with an LF construct can becompared with that of control (e.g., transformed host cells which aretransfected with a vector without insert). Host cells expressing afunctional LF would exhibit an increase in growth factor and serumdependence compared to control.

Tumor Specific Markers Levels

[0101] Tumor cells release an increased amount of certain factors(hereinafter “tumor specific markers”) than their normal counterparts.For example, plasminogen activator (PA) is released from human glioma ata higher level than from normal brain cells (see, e.g., Gullino,Angiogenesis, tumor vascularization, and potential interference withtumor growth. In Mihich (ed.): “Biological Responses in Cancer.” NewYork, Academic Press, pp. 178-184 (1985)). Similarly, Tumor angiogenesisfactor (TAF) is released at a higher level in tumor cells than theirnormal counterparts. See, e.g., Folkman, Angiogenesis and cancer, SemCancer Biol. (1992)).

[0102] Tumor specific markers can be assayed for to identify LFconstructs, which when expressed, decrease the level of release of thesemarkers from host cells. Typically, transformed or tumorigenic hostcells are used. Expression of the LF gene in these host cells wouldreduce or eliminate the release of tumor specific markers from thesecells. Therefore, this assay can be used to identify LF constructs fortreatment of cancer.

[0103] Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305-312 (1980);Gulino, Angiogenesis, tumor vascularization, and potential interferencewith tumor growth. In Mihich, E. (ed): “Biological Responses in Cancer.”New York, Plenum (1985); Freshney Anticancer Res. 5:111-130 (1985).

[0104] Invasiveness into Matrigel

[0105] The degree of invasiveness into Matrigel or some otherextracellular matrix constituent can be used as an assay to identify LFconstructs which are capable of inhibiting abnormal cell proliferationand tumor growth. Tumor cells exhibit a good correlation betweenmalignancy and invasiveness of cells into Matrigel or some otherextracellular matrix constituent. In this assay, tumorigenic cells aretypically used as host cells. Expression of an LF gene in these hostcells would decrease invasiveness of the host cells. Therefore,functional LF constructs can be identified by measuring changes in thelevel of invasiveness between the host cells before and after theintroduction of LF constructs. If an LF construct functions as a cancertherapeutic, its expression in tumorigenic host cells would decreaseinvasiveness.

[0106] Techniques described in Freshney (1994), supra, can be used.Briefly, the level of invasion of host cells can be measured by usingfilters coated with Matrigel or some other extracellular matrixconstituent. Penetration into the gel, or through to the distal side ofthe filter, is rated as invasiveness, and rated histologically by numberof cells and distance moved, or by prelabeling the cells with ¹²⁵I andcounting the radioactivity on the distal side of the filter or bottom ofthe dish. See, e.g., Freshney (1984), supra.

Tumor Growth in vivo

[0107] Effects of LF on cell growth can be tested in immune-suppressedmice. Various immune-suppressed or immune-deficient host animals can beused in these assays. For example, genetically athymic “nude” mouse(see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), aSCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g.,Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer41:52 (1980)) can be used as a host. Transplantable tumor cells(typically about 10⁶ cells) injected into isogenic hosts will produceinvasive tumors in a high proportions of cases, while normal cells ofsimilar origin will not. In hosts which developed invasive tumors, cellsexpressing an LF construct are injected subcutaneously. After a suitablelength of time, preferably 4-8 weeks, tumor growth is measured (e.g., byvolume or by its two largest dimensions) and compared to the control.Tumors that have statistically significant reduction (using, e.g.,Student's T test) are said to have inhibited growth. Using reduction oftumor size as an assay, functional LF constructs which are capable ofinhibiting abnormal cell proliferation can be identified.

[0108] B. In vitro Assays for Identification of LF Modulators andMimetics

[0109] LF homologues, modulators and mimetics can also be identifiedusing in vitro assays. Such assays are conveniently used for highthroughput screening of modulators and mimetics. For the in vitro assaysof the invention, recombinant or naturally occurring LF, PA, and MAPKKcan be used. For example, recombinant LF can be used in combination witha cell extract that has an activated MAPK pathway, e.g., a Mos-activatedoocyte lysate, or a Ras/Raf transformed NIH3T3 cell lysate, or any ofthe other cells described above. In such assays, direct cleavage ofMAPKK can be detected, or phosphorylation of MAPKK substrates can beexamined. Alternatively, recombinant or naturally occurring MAPKK and LFor LF homologues or mimetics are incubated together under standardreaction conditions, for direct detection of MAPKK cleavage. In anotherassay, recombinant or naturally occurring MAPKK and LF or an LFhomologue or mimetic are incubated with an MAPKK substrate such as MBPand/or MAPK, and phosphorylation of the substrate is examined.

[0110] The mimetics or modulators are added to the in vitro assays intest concentrations, by any suitable means. Typically, the modulators ormimetics are added to the assays in aqueous solutions, or in organicsolvents such as DMSO.

[0111] In one assay, oocyte extracts are examined for inhibition ofMos-activation of the MAPK pathway. Purified (recombinant or naturallyoccurring) Mos is added to the extract, along with LF or an LF mimetic,and/or a modulator, under suitable reaction conditions (see, e.g.,Example 1). LF modulator or mimetic is examined by direct detect ofMAPKK cleavage, using ELISA or western blots, as described above, or byexamining phosphorylation of MAPKK substrates such as MAPK, using ELISAor western blots, as described above.

[0112] In another assay, extracts from cells with activated MAPKpathways are examined. Purified (recombinant or naturally occurring) LFor LF mimetic, and optionally an LF modulator, are added to the extract.LF modulator or mimetic activity is examined by direct detect of MAPKKcleavage, using ELISA or western blots, as described above, or byexamining phosphorylation of MAPKK substrates such as MAPK, using ELISAor western blots, as described above.

[0113] In vitro assays can also be performed without cell extracts, bydirect incubation of LF or an LF mimetic and MAPKK, or by incubation ofLF or an LF mimetic and MAPKK with an MAPKK substrate. LF modulators areoptionally added to such assays. After LF or an LF homologue or mimeticand MAPKK are incubated together under suitable reaction conditions(see, e.g., Example IV). MAPKK cleavage is detected by using ELISA,western blots, or direct staining of SDS-PAGE gels. MAPKK cleavage canbe examined by using antibodies that specifically detect the truncatedMAPKK, or by using antibodies to the C-terminus of MAPKK and detectingaltered electrophoretic mobility. Altered electrophoretic mobility canalso be detected by direct staining of gels.

[0114] Phosphorylation of an MAPKK substrate can also be used as anassay for LF activity and MAPKK cleavage. For example, LF or an LFmimetic, MAPKK, MAPK, and myelin basic protein (MBP) are incubatedtogether in a kinase buffer, optionally with an LF modulator (see, e.g.,Example III). Phosphorylation of either MAPK or MBP can be detected bywestern blot or ELISA with specific antibodies to phosphorylated MAPK orMBP protein. Alternatively, γ-³²P-ATP can be added to the kinasereaction, and direct labeling of the proteins can be examined byelectrophoresis and autoradiography.

[0115] C. LF Modulators, Homologues, and Mimetics

[0116] New chemical or recombinant LF mimetics, homologues, andmodulators are generated by identifying compounds with LF MAPKK proteaseactivity or the ability to modulate LF MAPKK protease activity, usingthe assays described above. These compounds are often referred to aslead compounds. Once a lead compound is identified, variants aretypically created and evaluated for use as a therapeutic agent. Anexample of an LF mimetic is a small peptide containing the LF MAPKKprotease active site, or a small chemical molecule that has the samechemical structure as the LF active site. An example of an LF homologueis a naturally-occurring or recombinant variant of LF that has increasedstability or increased activity. An example of an LF modulator is asmall chemical or peptide that inhibits or activates LF MAPKK proteaseactivity.

[0117] A wide variety of LF homologues can be tested for LF MAPKKprotease activity, e.g., conservative modifications, truncations,targeted fusion proteins, etc. Such molecules are typically isolatedfrom naturally occurring strains of B. anthracis, or made using standardrecombinant technology, described below, or designed by computerassisted drug design, described below. For example, the LF MAPKKprotease active site can be identified using computer assisted drugdesign, by site-directed mutagenesis of conserved LF domains, byscreening nested deletions or by screening linker scanner deletions ofLF. The LF active site and conserved domains can also be identified bycomparing the amino acid sequences of LF alleles. LF homologues can thenbe designed that include the essential components of the active site andare conservatively modified in other regions. Standard recombinanttechniques are typically used to make LF homologues, e.g., site-directedmutagenesis, random mutagenesis, nested deletions, linker scannerdeletions, truncations, fusions, isolation of alleles from different B.anthracis strains, etc.

[0118] Combinatorial libraries also provide a source of potential LFhomologues, modulators, and mimetics, particularly mimetics andmodulators. In one embodiment, a library containing a large number ofpotential therapeutic compounds (candidate compounds) is provided. Such“combinatorial chemical libraries” are then screened in one or more LFMAPKK protease activity assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity. The compounds thus identified canserve as conventional “lead compounds” or can themselves be used aspotential or actual therapeutics.

[0119] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks called amino acids in every possible way for a given compoundlength (i.e., the number of amino acids in a polypeptide compound).Millions of chemical compounds can be synthesized through suchcombinatorial mixing of chemical building blocks.

[0120] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991); Houghton et al., Nature 354:84-88 (1991)). Peptide synthesis isby no means the only approach envisioned and intended for use with thepresent invention. Other chemistries for generating chemical diversitylibraries can also be used. Such chemistries include, but are notlimited to: peptoids (PCT Publication No. WO 91/19735), encoded peptides(PCT Publication WO 93/20242), random bio-oligomers (PCT Publication No.WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with a β-D-glucose scaffolding (Hirschmann et al., J.Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses ofsmall compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661(1994)), oligocarbamates (Cho et al., Science 261:1303 (1993)), and/orpeptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)),nucleic acid libraries, peptide nucleic acid libraries (see, e.g., U.S.Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522(1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries(see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514, and the like).

[0121] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).

[0122] A number of well known robotic systems have also been developedfor solution phase chemistries. These systems include automatedworkstations like the automated synthesis apparatus developed by TakedaChemical Industries, LTD. (Osaka, Japan) and many robotic systemsutilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.;Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manualsynthetic operations performed by a chemist. Any of the above devicesare suitable for use with the present invention. The nature andimplementation of modifications to these devices (if any) so that theycan operate as discussed herein will be apparent to persons skilled inthe relevant art. In addition, numerous combinatorial libraries arethemselves commercially available (see, e.g., ComGenex, Princeton, N.J.,Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow,RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md.,etc.).

[0123] D. High Throughput Methodology

[0124] Any of the assays for compounds that modulate or mimic LF MAPKKprotease activity, described herein, are amenable for use in highthroughput screening. High throughput assays for the activity of aparticular product, e.g., LF or LF mimetics or homologues, are wellknown to those of skill in the art. In addition, high throughputscreening systems are commercially available (see, e.g., Zymark Corp.,Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthruput and rapid start up as well as a high degree of flexibility andcustomization. The manufacturers of such systems provide detailedprotocols the various high throughput. Thus, for example, Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

[0125] Such high throughput assays often incorporate solid substratessuch as a membrane (e.g., nitrocellulose or nylon), a microtiter dish(e.g., PVC, polypropylene, or polystyrene), a test tube (glass orplastic), a dipstick (e.g., glass, PVC, polypropylene, polystyrene,latex, and the like), a microcentrifuge tube, or a glass, silica,plastic, metallic or polymer bead or other substrate such as paper.

[0126] Often in the assays of the invention, a molecule such as MAPKK islabeled with a detectable moiety. For example, in electrophoreticmobility assays, MAPKK can be labeled at the C-terminus or N-terminus(to observe cleavage by LF).

[0127] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with LF activity. Such detectable labels have beenwell-developed in the field of immunoassays and, in general, most anylabel useful in such methods can be applied to the present invention. Awide variety of labels may be used, with the choice of label dependingon sensitivity required, ease of conjugation with the compound,stability requirements, available instrumentation, and disposalprovisions.

[0128] Suitable labels are any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcolorimetric labels such as colloidal gold or colored glass or plasticbeads (e.g., polystyrene, polypropylene, latex, etc.).

[0129] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art.Non-radioactive labels are often attached by indirect means. Forexample, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize the LF substrate, or secondary antibodies that recognizeanti-LF-substrates.

[0130] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

[0131] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0132] E. Computer Assisted Drug Design

[0133] Yet another assay for compounds that modulate LF MAPKK proteaseactivity involves computer assisted drug design, in which a computersystem is used to generate a three-dimensional structure of a proteinbased on the structural information encoded by the amino acid sequence.The three dimensional structure of the protein is then used to identifypotential ligands that bind to the protein, or to identify moleculesthat are mimetics of the protein of interest. For example, thethree-dimensional structure of LF can be used to identify LF mimeticsand modulators that bind to LF. The structure can also be used toidentify the LF protease active site. Similarly, the three-dimensionalstructure of MAPKK can be used to identify LF mimetics that bind toMAPKK, or LF modulators that bind to MAPKK, particularly near theN-terminus where LF cleaves MAPKK. In the computer system, the inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., MAPKK or LF. Theseregions are then used to identify ligands that bind to the protein ofinterest, or regions where LF interacts with MAPKK, or the regions whereMAPKK interacts with LF.

[0134] The three-dimensional structural model of the protein isgenerated by entering channel protein amino acid sequences of at least10 amino acid residues or corresponding nucleic acid sequences encodingLF or MAPKK into the computer system. The amino acid sequence representsthe primary sequence or subsequence of the protein, which encodes thestructural information of the protein. The sequence is entered into thecomputer system from computer keyboards, computer readable substratesthat include, but are not limited to, electronic storage media (e.g.,magnetic diskettes, tapes, cartridges, and chips), optical media (e.g.,CD ROM), information distributed by internet sites, and by RAM. Thethree-dimensional structural model of LF or MAPKK is then generated bythe interaction of the amino acid sequence and the computer system,using software known to those of skill in the art.

[0135] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary and tertiarystructure of LF or MAPKK. The software looks at certain parametersencoded by the primary sequence to generate the structural model. Theseparameters are referred to as “energy terms,” and primarily includeelectrostatic potentials, hydrophobic potentials, solvent accessiblesurfaces, and hydrogen bonding. Secondary energy terms include van derWaals potentials. Biological molecules form the structures that minimizethe energy terms in a cumulative fashion. The computer program istherefore using these terms encoded by the primary structure or aminoacid sequence to create the secondary structural model.

[0136] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can enter additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and its cellular location, e.g., cytoplasmic,surface, or nuclear. These variables along with the energy terms of thesecondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

[0137] Once the structure has been generated, potential ligand bindingregions are identified by the computer system. Three-dimensionalstructures for potential ligands are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential ligand is thencompared to that of the LF or MAPKK protein to identify ligands thatbind to LF or MAPKK. Binding affinity between the protein and ligands isdetermined using energy terms to determine which ligands have anenhanced probability of binding to the protein.

[0138] IV. How to Make Recombinant LF, PA, and MAPKK Proteins

[0139] As described above, naturally occurring or recombinant LF andMAPKK and homologues and mimetics thereof can be used in the assays ofthe invention. Recombinant LF and MAPKK are conveniently used for invitro assays. In addition, recombinant LF homologues and fusion proteinscan be prepared for testing as LF mimetics and potential therapeuticagents. The preparation of recombinant LF, MAPKKs and PA is describedbelow, as well as methods for isolating naturally occurring proteins.

[0140] A. General Recombinant DNA Methods

[0141] Often, recombinant proteins are used in the assays of the presentinvention, e.g., recombinant LF, PA, and MAPKK, mutants thereof, andfunctional equivalents. For producing recombinant proteins, thisinvention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

[0142] For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). These are estimates derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

[0143] Oligonucleotides that are not commercially available can bechemically synthesized according to the solid phase phosphoramiditetriester method first described by Beaucage & Caruthers, TetrahedronLetts. 22:1859-1862 (1981), using an automated synthesizer, as describedin Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984).Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255:137-149 (1983).

[0144] The sequence of the cloned genes and synthetic oligonucleotidescan be verified after cloning using, e.g., the chain termination methodfor sequencing double-stranded templates of Wallace et al., Gene16:21-26 (1981).

[0145] B. Cloning Methods for the Isolation of Nucleotide SequencesEncoding LF, MAPKK, and PA

[0146] In general, the nucleic acid sequences encoding LF, MAPKK, PA,and related nucleic acid sequence homologues are cloned from cDNA andgenomic DNA libraries or isolated using amplification techniques witholigonucleotide primers. For example, LF and PA sequences are typicallyisolated from B. anthracis nucleic acid (genomic or cDNA) libraries,while genes for MAPKKs (e.g., MAPKK1, MAPKK2, etc.) can be cloned frommammalian libraries, preferably human libraries. For example, MAPKKssequences can be isolated from sarcoma libraries with an activated MAPKpathway.

[0147] Amplification techniques using primers can also be used toamplify and isolate LF, PA, and MAPKK from DNA or RNA (see U.S. Pat.Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)). Methods such as polymerasechain reaction (PCR) and ligase chain reaction (LCR) can be used toamplify nucleic acid sequences of LF directly from mRNA, from cDNA, fromgenomic libraries or cDNA libraries, and from plasmids. Degenerateoligonucleotides can be designed to amplify homologues. For example, thefollowing primers can also be used to amplify a sequence of LF: 5′ CCTAAG GGC ACA GCA AAG AAT GAG 3′ (SEQ ID NO:1) and 5′ GTG TGG CGA AAG TGGTGG TC 3′(SEQ ID nO:2). These primers can be used, e.g., to amplify aprobe of several hundred nucleotides, which is then used to screen ahuman library for full-length LF. Alternatively, the nucleic acid for LFcan be directly amplified. Similar procedures can be used to isolatesequences encoding PA and MAPKK.

[0148] Nucleic acids encoding LF, PA, and MAPKK can also be isolatedfrom expression libraries using antibodies as probes. Such polyclonal ormonoclonal antibodies can be raised using recombinant or naturallyoccurring LF, PA, or MAPKK as antigens.

[0149] Synthetic oligonucleotides can be used to construct recombinantLF genes for use as probes, for expression of protein, and forconstruction of polymorphic variants or mutants such as deletionmutants. This method is performed using a series of overlappingoligonucleotides usually 40-120 bp in length, representing both thesense and nonsense strands of the gene. These DNA fragments are thenannealed, ligated and cloned.

[0150] Polymorphic variants, alleles, and interspecies homologues thatare substantially identical to LF, PA, or MAPKK can be isolated usingLF, PA, and MAPKK nucleic acid probes and oligonucleotides understringent hybridization conditions, by screening libraries using probes,or using amplification techniques as described above. Alternatively,expression libraries can be used to clone polymorphic variants, alleles,and interspecies homologues, by detecting expressed homologuesimmunologically with antisera or purified antibodies, which alsorecognize and selectively bind to the homologue.

[0151] The gene encoding LF has been cloned and sequenced, and has beenassigned Genebank accession no. M29081 (Robertson & Leppla, Gene44:71-78 (1986); Bragg & Robertson, Gene 81:45-54 (1989); see also U.S.Pat. No. 5,591,631, U.S. Pat. No. 5,677,274; see generally Leppla,Anthrax Toxins, in Bacterial Toxins and Virulence Factors in Disease(Moss et al., eds., 1995)). The gene encoding PA has been cloned andsequenced, and assigned Genebank accession no. M22589 (Irvins & Welkos,Infect. Immun. 54:537-542 (1986); Welkos et al., Gene 69:287-300 (1988);see also U.S. Pat. No. 5,591,631, U.S. Pat. No. 5,677,274; see generallyLeppla, Anthrax Toxins, in Bacterial Toxins and Virulence Factors inDisease (Moss et al., eds., 1995)). The gene encoding, e.g., MAPKK1 andMAPKK2 have been cloned and sequenced. MAPKK1 has been assigned Genebankaccession no. L11284, and the accession no. for MAPKK2 is L11285 (see,e.g., Zheng & Guan, J. Biol. Chem. 268:11435-11439 (1993)).

[0152] The nucleic acids of interest are typically cloned intointermediate vectors before transformation into prokaryotic oreukaryotic cells for replication and/or expression. These intermediatevectors are typically prokaryote vectors, e.g., plasmids, or shuttlevectors, as described below.

[0153] C. Expression in Prokaryotes and Eukaryotes

[0154] To obtain high level expression of a cloned gene, such as thosecDNAs encoding LF, PA, and MAPKK, one typically subclones the nucleicacid into an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the LF protein are availablein, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

[0155] The promoter used to direct expression of a heterologous nucleicacid depends on the particular application and is not critical.Exemplary promoters include the SV40 early promoter, SV40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells, as well asprokaryotic promoters. The promoter is preferably positioned about thesame distance from the heterologous transcription start site as it isfrom the transcription start site in its natural setting. As is known inthe art, however, some variation in this distance can be accommodatedwithout loss of promoter function.

[0156] In addition to the promoter, the expression vector typicallycontains a transcription unit or expression cassette that contains allthe additional elements required for the expression of the nucleic acidin host cells. A typical expression cassette thus also contains signalsrequired for efficient polyadenylation of the transcript, ribosomebinding sites, and translation termination. The nucleic acid sequenceencoding the gene of choice may typically be linked to a cleavablesignal peptide sequence to promote secretion of the encoded protein bythe transformed cell. Such signal peptides would include, among others,the signal peptides from tissue plasminogen activator, insulin, andneuron growth factor, and juvenile hormone esterase of Heliothisvirescens. Additional elements of the cassette may include enhancersand, if genomic DNA is used as the structural gene, introns withfunctional splice donor and acceptor sites.

[0157] In addition to a promoter sequence, the expression cassetteshould also contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes.

[0158] Additional elements that are typically included in expressionvectors also include a replicon that functions in E. coli, a geneencoding antibiotic resistance to permit selection of bacteria thatharbor recombinant plasmids, unique restriction sites in nonessentialregions of the plasmid to allow insertion of eukaryotic sequences. Inaddition, some expression systems have markers that provide geneamplification such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. The particular antibiotic resistance genechosen is not critical, any of the many resistance genes known in theart are suitable. The prokaryotic sequences are preferably chosen suchthat they do not interfere with the replication of the DNA in eukaryoticcells, if necessary.

[0159] The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Other exemplary eukaryoticvectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculoviruspDSVE. Tags can also be added to recombinant proteins to provideconvenient methods of isolation, e.g., c-myc, or hexahistidine.

[0160] Standard transfection methods are used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofLF protein, which are then purified using standard techniques (see,e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques, e.g., calcium phosphate transfection,polybrene, protoplast fusion, electroporation, liposomes,microinjection, plasma vectors, viral vectors (see, e.g., Morrison, J.Baci. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

[0161] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe gene of choice, which is recovered from the culture using standardtechniques identified below.

[0162] V. Purification of LF, PA and MAPKK and Cellular Expression

[0163] Naturally occurring or recombinant LF, PA, and MAPKK can bepurified for use in the assays of the invention. Naturally occurring LFand PA are purified, e.g., from B. anthracis Sterne (Leppla, Productionand Purification of Anthrax Toxin, Methods Enymol. 165:103-116 (1988);Quinn et al., Biochem J. 252:753-758 (1988)). Naturally occurring MAPKKis purified from transformed mammalian cell lines. Recombinant LF, PA,and MAPKK are purified from any suitable expression system.

[0164] LF, PA, and MAPKK may be purified to substantial purity bystandard techniques, including selective precipitation with suchsubstances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra).

[0165] A number of procedures can be employed when recombinant proteinsare purified. For example, proteins having established molecularadhesion properties can be reversible fused to the protein of choice.With the appropriate ligand, the protein can be selectively adsorbed toa purification column and then freed from the column in a relativelypure form. The fused protein is then removed by enzymatic activity.Finally, the protein of choice can be purified using affinity orimmunoaffinity columns.

[0166] A. Purification of Protein from Recombinant Bacteria

[0167] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

[0168] Proteins expressed in bacteria may form insoluble aggregates(“inclusion bodies”). Several protocols are suitable for purification ofrecombinant inclusion bodies. For example, purification of inclusionbodies typically involves the extraction, separation and/or purificationof inclusion bodies by disruption of bacterial cells, e.g., byincubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂,1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysedusing 2-3 passages through a French Press, homogenized using a Polytron(Brinkman Instruments) or sonicated on ice. Alternate methods of lysingbacteria are apparent to those of skill in the art (see, e.g., Sambrooket al., supra; Ausubel et al., supra).

[0169] If necessary, the inclusion bodies are solubilized, and the lysedcell suspension is typically centrifuged to remove unwanted insolublematter. Proteins that formed the inclusion bodies may be renatured bydilution or dialysis with a compatible buffer. Suitable solventsinclude, but are not limited to urea (from about 4 M to about 8 M),formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Other suitable buffers areknown to those skilled in the art. The protein of choice is separatedfrom other bacterial proteins by standard separation techniques, e.g.,with Ni-NTA agarose resin.

[0170] Alternatively, it is possible to purify the recombinant proteinfrom bacteria periplasm. After lysis of the bacteria, when therecombinant protein is exported into the periplasm of the bacteria, theperiplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes, for cell lysis to occur. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

[0171] B. Standard Protein Separation Techniques for PurifyingRecombinant and Naturally Occurring Proteins

[0172] Solubility Fractionation

[0173] Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the protein of interest. The preferred salt is ammonium sulfate.Ammonium sulfate precipitates proteins by effectively reducing theamount of water in the protein mixture. Proteins then precipitate on thebasis of their solubility. The more hydrophobic a protein is, the morelikely it is to precipitate at lower ammonium sulfate concentrations. Atypical protocol includes adding saturated ammonium sulfate to a proteinsolution so that the resultant ammonium sulfate concentration is between20-30%. This concentration will precipitate the most hydrophobic ofproteins. The precipitate is then discarded (unless the protein ofinterest is hydrophobic) and ammonium sulfate is added to thesupernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

[0174] Size Differential Filtration

[0175] The molecular weight of the protein of choice can be used toisolated it from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

[0176] Column Chromatography

[0177] The protein of choice can also be separated from other proteinson the basis of its size, net surface charge, hydrophobicity, andaffinity for ligands. In addition, antibodies raised against recombinantor naturally occurring proteins can be conjugated to column matrices andthe proteins immunopurified. All of these methods are well known in theart. It will be apparent to one of skill that chromatographic techniquescan be performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech). For example, LF can be purifiedusing a PA63 heptamer affinity column (Singh et al., J. Biol. Chem.269:29039-29046 (1994)).

[0178] VI. Kits

[0179] The present invention also provides for kits for screening formodulators of LF. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: biologically active LF, reaction tubes,and instructions for testing LF activity. A wide variety of kits andcomponents can be prepared according to the present invention, dependingupon the intended user of the kit and the particular needs of the user.For example, the kit can be tailored for ex vivo or in vitroMos-activation of MAPK, in vitro phosphorylation of MBP, ex vivo or invitro cleavage of MAPKK and determination of electrophoretic mobility.

[0180] VII. Gene Therapy

[0181] The present invention provides the nucleic acids of LF and LFhomologues for the transfection of cells in vitro and in vivo. Thesenucleic acids can be inserted into any of a number of well known vectorsfor the transfection of target cells and organisms as described below.The nucleic acids are transfected into cells, ex vivo or in vivo,through the interaction of the vector and the target cell. The nucleicacids encoding LF and LF homologues, under the control of a promoter,then expresses an LF of the present invention, thereby providing atherapeutic reagent to a cancer cell.

[0182] Such gene therapy procedures have been used to correct acquiredand inherited genetic defects, cancer, and viral infection in a numberof contexts. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies (for a review of gene therapy procedures, see Anderson,Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology (Doerfler & Böhmeds., 1995); and Yu et al., Gene Therapy 1: 13-26 (1994)).

[0183] Delivery of the gene or genetic material into the cell is thefirst critical step in gene therapy treatment of disease. A large numberof delivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

[0184] Methods of non-viral delivery of nucleic acids includelipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in, e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787;and U.S. Pat. No. 4,897,355) and lipofection reagents are soldcommercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutrallipids that are suitable for efficient receptor-recognition lipofectionof polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

[0185] The preparation of lipid:nucleic acid complexes, includingtargeted liposomes such as immunolipid complexes, is well known to oneof skill in the art (see, e.g., Crystal, Science 270:404410 (1995);Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al.,Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem.5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad etal., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183,4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,4,837,028, and 4,946,787).

[0186] The use of RNA or DNA viral based systems for the delivery ofnucleic acids take advantage of highly evolved processes for targeting avirus to specific cells in the body and trafficking the viral payload tothe nucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

[0187] The tropism of a retrovirus can be altered by incorporatingforeign envelope proteins, expanding the potential target population oftarget cells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

[0188] In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

[0189] In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

[0190] pLASN and MPG-S are examples are retroviral vectors that havebeen used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995);Kohn et al., Nat. Med. 1: 1017-102 (1995); Malech et al., Proc. Natl.Acad. Sci. U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the firsttherapeutic vector used in a gene therapy trial. (Blaese et al., Science270:475480 (1995)). Transduction efficiencies of 50% or greater havebeen observed for MFG-S packaged vectors. (Ellem et al., ImmunolImmunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2(1997).

[0191] Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

[0192] Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiply types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

[0193] Packaging cells are used to form virus particles that are capableof infecting a host cell. Such cells include 293 cells, which packageadenovirus, and ø2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

[0194] In many gene therapy applications, it is desirable that the genetherapy vector be delivered with a high degree of specificity to aparticular tissue type. A viral vector is typically modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the viruses outer surface. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. U.S.A. 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human. heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other pairs of virus expressing a ligand fusion protein and targetcell expressing a receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., Fab or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences thought to favor uptakeby specific target cells.

[0195] Gene therapy vectors can be delivered in vivo by administrationto an individual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

[0196] Ex vivo cell transfection for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transfected cells into the hostorganism) is well known to those of skill in the art. In a preferredembodiment, cells are isolated from the subject organism, transfectedwith a nucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

[0197] In one embodiment, stem cells are used in ex vivo procedures forcell transfection and gene therapy. The advantage to using stem cells isthat they can be differentiated into other cell types in vitro, or canbe introduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-alpha and TNF-alpha are known (see Inaba et al., J.Exp. Med. 176:1693-1702 (1992)).

[0198] Stem cells are isolated for transduction and differentiationusing known methods. For example, stem cells are isolated from bonemarrow cells by panning the bone marrow cells with antibodies which bindunwanted cells, such as CD4+ and CD8+(T cells), CD45+(panb cells), GR-1(granulocytes), and lad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

[0199] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)containing therapeutic nucleic acids can be also administered directlyto the organism for transduction of cells in vivo. Alternatively, nakedDNA can be administered.

[0200] Administration is by any of the routes normally used forintroducing a molecule into ultimate contact with blood or tissue cells,as described below. The nucleic acids are administered in any suitablemanner, preferably with pharmaceutically acceptable carriers. Suitablemethods of administering such nucleic acids are available and well knownto those of skill in the art, and, although more than one route can beused to administer a particular composition, a particular route canoften provide a more immediate and more effective reaction than anotherroute (see Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989)). In particular, atleast six viral vector approaches are currently available for genetransfer in clinical trials, with retroviral vectors by far the mostfrequently used system. All of these viral vectors utilize approachesthat involve complementation of defective vectors by genes inserted intohelper cell lines to generate the transducing agent.

[0201] VIII. Pharmaceutical Compositions and Administration

[0202] LF and LF homologue nucleic acid and protein, PA protein, andmodulators and mimetics of LF can be administered directly to thepatient for inhibition of cancer, tumor, or precancer cells in vivo.Administration is by any of the routes normally used for introducing acompound into ultimate contact with the tissue to be treated. Thecompounds are administered in any suitable manner, preferably withpharmaceutically acceptable carriers. Suitable methods of administeringsuch compounds are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route. As describedabove, in one embodiment, LF and LF homologues are administered to acell in the presence of PA or a targeted PA fusion protein.

[0203] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985)).

[0204] The compounds (nucleic acids, proteins, and modulators), alone orin combination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

[0205] Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, by intravenous infusion,orally, topically, intraperitoneally, intravesically or intrathecally.The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

[0206] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular compound employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular compound or vector in a particularpatient

[0207] In determining the effective amount of the modulator to beadministered in the treatment or prophylaxis of cancer, the physicianevaluates circulating plasma levels of the modulator, modulatortoxicities, progression of the disease, and the production ofanti-modulator antibodies. In general, the dose equivalent of amodulator is from about 1 ng/kg to 10 mg/kg for a typical patient.Administration of compounds is well known to those of skill in the art(see, e.g., Bansinath et al., Neurochem Res. 18:1063-1066 (1993);Iwasaki et al., Jpn. J. Cancer Res. 88:861-866 (1997); Tabrizi-Rad etal., Br. J. Pharmacol. 111:394-396 (1994)).

[0208] For administration, modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

[0209] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0210] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

[0211] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar results.

Example 1 In vitro and ex vivo Inhibition of Mos Activation ofMaturation Promoting Factor

[0212] The activity of anthrax lethal toxin as an inhibitor of the MAPKsignal transduction pathway can be examined in Xenopus oocyte extracts.Activation of the MAPK pathway by newly synthesized Mos is essential forthe activation of maturation promoting factor (i.e., cyclin B/p34^(cdc2)kinase) and the resumption of meiosis (maturation) in oocytes (seeMurakami et al., Methods in Enzymology 283:584-600 (Dunphy, ed., 1991)).Accordingly, the effect of lethal factor was assayed by examininginhibition of Mos activation of oocyte maturation, using oocyte extractsand direct injection of LF into oocytes.

[0213] a. Ex vivo Inhibition

[0214] For the ex vivo assays, Xenopus oocytes were isolated,defolliculated, injected, and induced to mature with progesterone asdescribed previously (Duesbery et al., Proc. Natl. Acad. Sci. USA94:9165-9170 (1997)). LF and PA were purified from culture supernatantsof B. anthracis Sterne, a strain that produces PA, LF, and EF, usingmethods described previously (Leppla, in Methods in Enzymology165:103-116 (Harshman, ed., 1988)). PA and LF were either added tooocyte culture medium, or LF was directly injected into oocytes.

[0215] Addition of FPLC-purified PA and LF, prepared from B. anthracisSterne, to oocyte culture medium had no effect on oocyte maturation. Bycontrast, injection of LF into oocytes potently inhibitedprogesterone-induced maturation (Table 1). Injection of as little as 1ng LF inhibited maturation by 50% as judged by assay of germinal vesicle(i.e., nuclear envelope) breakdown (GVBD), and GVBD was completelyinhibited by 10 ng LF. To assay GVBD breakdown, oocytes were fixed informalin fixative and manually dissected with a scalpel to determinewhether the germinal vesicle was still intact (Duesbery & Masui, Dev.Genes Evol. 206:110-124 (1996)). The injection of the inactive LFmutant, LF E687C, had no effect upon GVBD (Table 1). Preparations of LFfrom strains of B. anthracis deficient in the production of EF alsoblocked oocyte maturation (Table 1). The inhibitory effects of LF uponprogesterone-induced GVBD were reversible since the subsequent injectionof A90 cyclin B, a truncated, non-degradable form of cyclin B (Glotzeret al., Nature 349:132-138 (1991)) could induce GVBD (Table 1). TABLE 1LF blocks oocyte maturation progesterone Material injected treatmentGVBD (#frogs used) none − 0/128 (7)^(†) none + 143/158 (7)^(†) injectionbuffers + 61/75 (4)^(†)  1 ng LF + 24/52 (3)^(†) 10 ng LF + 0/50 (3)^(†)40 ng LF + 0/75 (4)^(†) 40 ng LF(E687C) + 57/73 (4)^(†) none − 0/99(3)^(¶) none + 108/113 (3)^(¶) 40 ug LF ^(∥) + 0/75 (3)^(¶) 24 ng Δ90cyclin − 43/43 (2)^(¶) 40 ng LF ^(∥,) − 75/75 (3)^(¶) 24 ng Δ90 cyclin

[0216] b. In vitro Inhibition

[0217] The oocyte extract assay was performed as follows. LF or LFmutant E687C (4 μg from a 1 mg/mL stock) was added to 40 μl oocytelysate (Shibuya et al., EMBO J. 11:3963-3975 (1992)). LF E867C is aninactive LF mutant described in Klimpel et al., Mol. Microbiol.13:1093-1100 (1994). Lysates were activated 0.5 hr later by the additionof 2.6 μg maltose binding protein-Mos fusion protein (0.75 mg/mL stock).Mos with wild type activity was purified from bacteria as a malEmos^(Xe) fusion product (Yew et al., Nature 355:649-652 (1992)).Aliquots were taken at 1 hr intervals and frozen for later analysis bySDS-PAGE and western blotting (Duesbery et al., Proc. Natl. Acad. Sci.USA 94:9165-9170 (1997)). Blots were probed with antibodies raisedagainst phosphorylated MAPK (PO₄-MAPK) (New England Laboratories, 1:1000), MAPK (Zymed clone ERK-7D8, 1:1000), the C-terminus of MAPPKL(MAPKK1 (Ct)) (Upstate Biotechnology, 1:500), or the N-terminus ofMAPPKL (MAPKK1 (Nt)) (Upstate Biotechnology, 1:500) and visualized bychemiluminescence with HRP-conjugated secondary antibodies.

[0218] The addition of LF, but not LF E687C, inhibited Mos-inducedactivation of MAPK in oocyte lysates, suggesting that oocytes are unableto mature in the presence of LF due to a failure in MAPK activation.When western blots of these lysates were probed with antibodies to theC-terminus of MAPKK1, antigen was detected throughout the incubation(albeit at reduced levels when compared to control lysates), whereas, ifprobed with antibodies to the N-terminus of MAPKK1, the antigen was notdetected at any time after the addition of LF.

[0219] These results revealed that LF modifies MAPKK1, rendering theprotein undetectable by antibodies raised against its N-terminus.Indeed, in the presence of LF, the mobility of MAPKK1 observed with theC-terminal antibody increased slightly, suggesting that MAPKK1 wasproteolytically modified.

Example II Activity of LF in Cells that have an Activated MAPK Pathway

[0220] The effects of LF were tested upon tumor-derived NIH3T3 (490)cells expressing an effector domain mutant form of the human V12HaRasoncogene (V12-S35 Hras). This oncogenic mutant of Ras retainsconstitutive activation of the Raf-MAPKK-MAPK pathway, but is defectivein other effector functions (White et al., Cell 80:533-541 (1995)).Cells transformed with this Ras mutant are tumorigenic, and derivedtumors display high levels of MAPK (ERK 1/2) activity. These cells wereused instead of wild type V12HRas transformed cells to eliminate thepossibility of MEK-independent pathways contributing to ERK 1/2activity.

[0221] The NIH3T3 cells expressing an effector domain mutant form of thehuman V12HaRas oncogene (V12-S35 Hras) were grown in 6-well plates toapproximately 70% confluence in DMEM+10% FBS Cells were then incubatedin fresh DMEM+10%FBS containing 1 g/mL of PA for 10 min. Control media,LF, or, LF E687C was then added directly to the cells at a finalconcentration of 0.1 μg/mL. Cells were lysed (at 20 min., 1 hr. or 3hrs.) in lysis buffer (20 mM Pipes, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1.5mM MgCl₂, 1% SDS, 10 μg/mL aprotonin and leupeptin, 1 mM PMSF, 1 mMsodium orthovanadate) and cell lysates were clarified by centrifugation(15,000 g for 15 min., 4° C.). Protein concentrations in clarified celllysates were determined using the Pierce BCA protein determination kitand samples (10 μg) were analyzed by SDS-PAGE and western blotting asdescribed above with the exception that antibodies raised againstphosphorylated MAPK were obtained from Promega (1:15,000).

[0222] The addition of PA and LF, but not LF E687C, to these cellsinhibited MAPK activation. This inhibition was also accompanied by anincrease in the electrophoretic mobility of MAPKK1 observed with theC-terminal antibody, as well as a loss of MAPKK1 epitopes observed withthe N-terminal antibody, further demonstrating that LF proteolyticallymodifies MAPKK1.

Example III In vitro Assay for LF Activity Using MBP Phosphorylation

[0223] The effects of LF upon MAPK activation were directly demonstratedby assaying, in vitro, myelin basic protein (MBP) phosphorylation in thepresence of MAPKK1 and MAPK (ERK 2).

[0224] His-tagged MAPKK1, possessing endogenous activity (0.25 μg from a0.1 mg/mL stock prepared from bacterial lysates) was diluted in 16 μLassay buffer (composed of (1) 8 μL assay dilution buffer (ADB, UpstateBiotechnology; 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EGTA,1 mM sodium orthovanadate, 1 mM dithiothreitol); and (2) 8 μL inhibitorcocktail (Upstate Biotechnology; 20 mM PKC inhibitor peptide, 2 mMprotein kinase A inhibitor peptide, and 20 mM compound R24571 in ADB));and incubated for 15 min. at 30° C. in the presence or absence of 0.25μg LF or LF E687C.

[0225] After incubation, samples were added to 11 μL kinase buffer,composed of: 0.35 μg Erk2 (from a 0.35 mg/ml stock); (His)₆-tagged MAPK(expressed in bacterial cells obtained from Drs. Cobb and Robbins, andpurified as described by Robbins, et al., J. Biol Chem. 268:5097-5106(1993); 10 μg myelin basic protein (MBP) (Upstate Biotechnology 5mg/ml); and 8 μL Mg/ATP solution ((γ-³²P)-ATP (Amersham; 10 mCi/ml, 3000mCi/mmol) diluted 1:9 in 0.5 mM ATP, 75 MM MgCl₂ in ADB). After 15 min.incubation at 30° C., samples were separated by SDS-PAGE upon 14% gelsand processed for autoradiography.

[0226] The addition of LF, but not LF E687C, prevented MBPphosphorylation. Thus, as seen ex vivo, LF directly inhibits MAPKactivation in vitro.

[0227] To exclude the possibility that contaminants in the LFpreparation may be responsible for the inhibition of MBPphosphorylation, LF was adsorbed to the PA63 heptamer, to which ittightly binds (Singh et al., J. Biol. Chem. 269:29039-29046 (1995)), andre-purified by column chromatography, as follows.

[0228] LF previously purified by hydroxylapatite and ion exchangechromatography was re-chromatographed on a MonoQ HR5/5 column in thepresence and absence of the PA63 heptamer. The MonoQ column was elutedwith a gradient of NaCl in 10 mM CHES, 0.06% aminoethanol, pH 9.0. Thesamples applied to the columns were 250 μg LF, 250 μg PA63, and 250 μgLF+350 μg PA63. Fractions were pooled and assayed for inhibition ofMAPKK1 activity as described above. Lane 1, LF alone (fractions 18-23);lanes 2, 3 and 4, PA63 alone (fractions 18-23 (lane 2), 24-29 (lane 3),and 27-28 (lane 4)); lanes 5, 6 and 7, PA63 & LF (fractions 18-23 (lane5), 24-29 (lane 6), and 27-28 (lane 7)). Western blotting confirmed thatall the LF protein bound to the PA63 heptamer and eluted in peaks 6 & 7.

[0229] In all cases, the inhibition of MBP phosphorylation was found toco-elute with LF. MAPKK1 inhibitory activity therefore co-migrates withLF repurified by adsorption to PA63.

Example IV Assay in vitro for LF MAPKK Activity Using Changes in MAPKK1Electrophoretic Mobility

[0230] Direct testing for LF cleavage of MAPKK1 was performed byexamining the increase in electrophoretic mobility of MAPKK1 and thedisappearance of N-terminal epitopes in vitro. This assay was performedusing a His₆-tagged MAPKK1 fusion protein produced in bacteria.

[0231] The assay was performed as follows. His-tagged MAPKK1 (0.1 μg)was incubated in 16 μl assay buffer, as described above, in the presenceof 1 μg LF E687C (lanes 1-3) or 1 μg LF (lanes 4-6). Samples werewithdrawn at 0, 10, or 20 min. and analyzed by SDS-PAGE and westernblotting with antibodies raised against the C-terminus of MAPKK1.

[0232] This experiment demonstrated that, within seconds of LF addition,the apparent M_(r) of MAPKK1 decreased by approximately 6-8 kDa.Furthermore, cleavage by LF is enzymatic, since LF proteolysis of MAPKK1was observed within 15 min with as little as 2 ng LF per 200 ng MAPKK1(approximately 1 mol LF: 400 mol MAPKK1).

[0233] To demonstrate that cleavage by LF is enzymatic, His₆-MAPKK1 (0.2μg) was incubated as described above in the presence of 2 μg LF E687C(lane 1) or LF (lanes 2-6), which had been serially diluted in ADB (2 μgto 0.2 ng). Aliquots were withdrawn at the 15 and 30 min. time pointsand analyzed by SDS-PAGE and western blotting with antibodies raisedagainst the C-terminus of MAPKK1.

[0234] Since the MAPKK1 used in these analyses is His-6-tagged at theN-terminus (Mansour et al., Cell Growth and Differ. 7:243-250 (1996)),the actual decrease in the M_(r) of MAPKK1 is approximately 5 kDa less(Mansour et al., Cell Growth and Differ. 7:243-250 (1996)), suggestingthat LF cleaves MAPKK1 in the first 30 amino acids.

Example V Determination of MAPKK Cleavage Site for LF

[0235] To determine where LF cleaves MAPKK1, previously preparedN-terminal deletion mutants of MAPKK1 (Mansour et al., Science265:966-970 (1994)) were assayed for their ability to serve assubstrates for LF, as follows.

[0236] His-tagged MAPKK1 deletion mutants (0.1 μg) were isolated frombacterial lysates as described (Mansour et al., Science 265:966-970(1994), were incubated in assay buffer as described in the presence orabsence of LF (1 μg) for 15 min. at 30° C., and were analyzed bySDS-PAGE and western blotting as described above. MAPKK1 deletionmutants ΔN1 and ΔN2 were completely resistant to proteolysis, whereasΔN3, Δ4, and Δ 6 were cleaved. ΔN5 showed partial resistance toproteolysis, suggesting that structural modifications in this constructmay partially hinder LF activity.

[0237] These analyses showed that ΔN3 (32-51), ΔN4 (44-51), ΔN5 (3843),and ΔN6 (32-37) were susceptible to LF proteolysis whereas ΔN1 (1-32)and ΔN2 (1-52) were not. Thus, the N-terminal 32 amino acids areessential for cleavage and/or binding of MAPKK1 by LF.

Example VI Identification of LF Cleavage Site on MAPKK1

[0238] To determine the exact site of LF cleavage of MAPKK1, N-terminalsequence analysis of the larger MAPKK1 proteolytic fragment wasperformed as described above.

[0239] MAPKK1 deletion mutants (0.1 μg) were incubated with LF (0.2 μg)as described for 30 min at 30° C., after which samples were separated bySDS-PAGE and blotted onto PVDF membrane in CAPS transfer buffer(3-[cyclohexylaminol-1 propanesulfonic acid (10 mM, pH 11), 10%methanol) at 300 mA constant current for 30 min. Following transfer,membranes were quickly stained with Ponceau S solution (0.1% Ponceau S,5% acetic acid) and rinsed with distilled, deionized water.

[0240] For sequence analysis, the appropriate band was cut from themembrane and subjected to automated Edman degradation in an Applied Biosystems 477A gas-phase sequencer and phenylthiohydantoin derivativeswere identified on line with a 120 phenylthiohydantoin analyzer.

[0241] After sequence analysis, the amino acid sequence IQLNPAPDG wasidentified, which corresponds to amino acids 8-16 of MAPKK1. Thus, LFcleaves MAPKK1 between amino acids 7 and 8, resulting in the loss of theN-terminal seven residues (PKKKPTP). Consistent with the ex vivo and invitro assays, previous analysis of MAPKK1 deletion mutants has indicatedthat mutants with deletions of the N-terminal 32 amino acids possessless activity than wild-type MAPKK1 (Mansour et al., Biochemistry35:15529-15536 (1996)). In addition, the N-terminal 32 amino acids ofMAPKK1 contain a MAPK binding site (Fukuda et al., EMBO J. 16:1901-1908(1997)), suggesting that LF may prevent the association of MAPKK1 withits substrate. These results demonstrate that the seven N-terminalresidues of MAPKK1 are essential for its activity. MAPKK1 prolines 5 and7 were each separately mutated to an alanine in two MAPKK1 mutants. Bothmutants were resistant to LF cleavage, indicating that these prolineresidues may be an important component of the cleavage site.

[0242] As observed for MAPKK1, the electrophoretic mobility of MAPKK2increased with LF treatment. Sequence analysis was performed for theN-terminus of the larger MAPKK2 proteolytic fragment. LF cleaved MAPKK2between residues 9 and 10, resulting in the loss of N-terminal residues1 to 9 (LARRKPVLP). LF also cleaves MAPKK3.

[0243] The results presented above show that frog, mouse, and humanMAPKK1, as well as human MAPKK2 and MAPKK3, are all substrates of LF(see FIG. 1).

Example VII Effects of LF and PA on V12-H-ras Transformed Cells

[0244] NIH3T3 cells transformed with V12H-ras exhibit characteristicstypical of transformed cells, including a distinct morphology, anability to form foci very rapidly, a diffuse actin staining pattern, arapid proliferation rate, and an ability to grow independent ofanchorage to a substrate. As described infra, LF and PA can reverse eachof these properties of V12H-ras transformed cells, restoring to thecells properties typical of normal, non-transformed cells.

[0245] a. Morphological Changes

[0246] Cells transformed with human H-ras (V12) have a distinctappearance characterized by long, spindle-shaped cells and an ability toform foci very rapidly. To assess the ability of PA and LF to reversethese morphological features, NIH3T3 (490) cells expressing human H-ras(V12) protein were grown in DMEM supplemented with 10% fetal bovineserum, 2% penicillin/streptomycin, and maintained at 37° C. in ahumidified atmosphere of 10% CO₂, and with or without the presence of PAand LF. After 24 hours in the presence of PA and LF, the transformedcells lost the distinct appearance described above and assumed aflatter, larger appearance typical of non-transformed cells. Further,cells grown in the presence of PA and LF failed to form foci. Incontrast, cells grown in the presence of PA alone, or PA and LF E687C,failed to undergo any morphological changes.

[0247] Another characteristic feature of H-ras (V12) transformed cellsis a diffuse pattern of actin staining. To assess whether PA and LF wereable to restore a normal, non-transformed pattern of actin distributionto the transformed NIH3T3 cells, the cells were stained for actin andexamined using a confocal microscope. Cells were grown on 4 well glassslides (Nunc, IL), washed twice with PBS, and fixed with methanol for 15minutes at 4° C. After the methanol was removed, cells were air-driedand then rehydrated with PBS for 5 minutes. Cells were then post-fixedwith 4% formalin for 15 minutes, washed with PBS and permeabilized with1% NP-40 in PBS for 5 minutes. After rinsing, cells were incubated withblocking solution antibody (PBS containing 10% normal goat serum, 3%BSA, 0.1% Tween-20) for 1 hour at 37° C. The cells were then incubatedwith mouse monoclonal anti-actin (Sigma, clone KJ43A; 1:250) (inblocking solution) for 1 hour at room temperature. The antigen-antibodycomplexes were detected with Texas red conjugated anti-mouse IgGantibody (Molecular probes; 1:250) for 30 minutes at room temperatureand rinsed three times. Coverslips were mounted in aqueousnon-fluorescing medium containing 1 mg/ml Hoechst 33342. Slides werethen examined by confocal laser scanning microscopy.

[0248] Transformed cells grown in the absence of LF and PA displayed, asdescribed above, a diffuse pattern of actin staining. In contrast, intransformed cells treated with PA and LF, the actin was organized into“stress fibers” typical of non-transformed cells. Transformed cellsgrown in the presence of PA alone, or PA and LF E687C, failed to showany changes in actin distribution.

[0249] b. Cell Cycle

[0250] To determine whether the presence of LF and PA can inhibit therapid proliferation characteristic of transformed cells, transformedcells were incubated, as described supra, in the presence or absence ofLT for 0-7 days. Analysis of the proliferation rate of the cellsdemonstrated that LT inhibited the proliferation of the cells. Todetermine the cell cycle stage affected by LT-mediated inhibition, thecells were analyzed for cell cycle distribution by flow cytometry usingthe Cellular DNA Flow Cytometric analysis reagent set (BoehringerMannheim, Ind.). At various sampling points, medium was collected andcombined with trypsinized cells and centrifuged at 2000 rpm for 5minutes in a Beckmann GS-6R centrifuge at 4° C. The resulting pellet waswashed once with cold Versene 1:5000 (Gibco-BRL, NY) and fixed in 70%ethanol for 30 minutes at 4° C. Cells were then spun down and rehydratedin 1 ml PBS (−Ca²⁺/Mg²⁺) at room temperature for 30 minutes, at whichpoint 10 ml FCS was added and cells were incubated for 30 additionalminutes. Cells were then spun down and incubated in 1 ml PBS(−Ca²⁺/Mg²⁺) including 5 ml RNAse (DNAse free) and 100 ml propidiumiodide at room temperature for 30 minutes. Samples were then subjectedto flow cytometric analysis. This analysis of the cells by fluorescenceactivated cell sorting indicated that treatment with LT caused the cellsto arrest at G1 phase of the cell cycle.

[0251] c. Anchorage-Independent Growth

[0252] Another hallmark of the transformed cell is its ability to growindependent of its anchorage to a substrate and to invade other tissues.These characteristics may be assayed in vitro by monitoring the abilityof single cell suspensions to proliferate in soft agar or to ‘branch’while suspended in medium that mimics the extracellular environment ofthe cell, e.g., Matrigel. To assess the ability of LT to reverse theseproperties, ras-transformed NIH3T3 cells were grown in the presence orabsence of LT and assayed for the above-described properties.

[0253] Three-dimensional Matrigel invasion assays were performed asdescribed previously (Jeffers et al., Mol. Cell. Biol. 16:1115 (1996))with modification. Cells were collected by centrifugation afterincubation in PBS containing 0.2 g/L EDTA (Versene, Gibco BRL) at 37° C.for 0.5 hours. Approximately 2.5×10⁴ cells in a volume of 62.5 ml DMEM,10% fetal bovine serum, were mixed with an equal volume of non-dilutedGFR-Matrigel (see, e.g., Martin et al,. Hum. Reprod. 13:1645 (1998))supplemented with or without PA (1 mg/ml) or LF (E687C) (0.01-1 mg/ml).The cell suspension was then added to a 96-well culture plate andincubated 0.5 hours at 37° C., 10% CO₂, to solidify. DMEM containing 10%calf serum was then layered overtop and the cells were further incubatedat 37° C., 5% CO₂ for up to one week, during which cells were monitoreddaily. Each sample was assayed in triplicate in three separateexperiments. In these experiments, the presence of LT prevented thetransformed cells from undergoing branching morphogenesis in theMatrigel.

[0254] For soft agar colony formation assays, trypsinized cells werewashed with Ca²⁺/Mg²⁺-free PBS, resuspended at a concentration of 1×10⁴cells/ml in DMEM containing 10% calf serum, 0.5% (W/V) Noble agar (Difcolaboratories, MI), in the presence or absence of LT, and layered over a0.5 ml solid plug of DMEM containing 1% agar in 24 well plates. It wasobserved that cells grown in the absence of LT were capable ofproliferating in the agar, in contrast to cells grown in the presence ofLT, which were prevented from proliferating in the agar.

[0255] The above results demonstrate that LT can reverse multipleaspects of the transformed phenotype, including morphological features,proliferation, and anchorage-independent growth.

What is claimed is:
 1. An in vitro method for screening modulators oflethal factor (LF) mitogen activated protein kinase kinase (MAPKK)protease activity, the method comprising the steps of: (i) providing LFin an aqueous solution, wherein the LF has MAPKK protease activity inthe solution; (ii) contacting LF with substances suspected of having theability to modulate MAPKK protease activity; and (iii) assaying for thelevel of LF MAPKK protease activity.
 2. The method of claim 1, whereinthe LF is recombinant.
 3. The method of claim 1, wherein the step ofassaying comprises a Mos-induced activation of MAPK assay in a Xenopusoocyte lysate.
 4. The method of claim 1, wherein the step of assayingcomprises an MAPKK1 or MAPKK2 mobility assay.
 5. The method of claim 4,wherein the MAPKK1 or MAPKK2 is recombinant.
 6. The method of claim 5,wherein the recombinant MAPKK1 or recombinant MAPKK2 is linked to adetectable moiety.
 7. The method of claim 1, wherein the step ofassaying comprises an myelin basic protein (MBP) phosphorylation assay.8. A kit for screening in vitro for modulators of lethal factor (LF)mitogen activated protein kinase kinase (MAPKK) protease activity, thekit comprising; (i) a container holding LF, wherein the LF has MAPKKprotease activity; and (ii) instructions for assaying for LF MAPKKprotease activity.
 9. A kit of claim 8, wherein the LF is recombinant.10. A in vivo method for screening modulators of lethal factor (LF)mitogen activated protein kinase kinase (MAPKK) protease activity, themethod comprising the steps of: (i) contacting a living cell with LF,wherein the LF has MAPKK protease activity; (ii) contacting the cellwith substances suspected of having the ability to modulate MAPKKprotease activity; and (iii) assaying for the level of LF MAPKK proteaseactivity.
 11. The method of claim 10, wherein the LF is recombinant. 12.The method of claim 10, wherein the step of contacting the cellcomprises transducing the cell with an expression vector encoding LF.13. The method of claim 10, wherein the step of contacting furthercomprises contacting a cell with LF in the presence of protectiveantigen (PA).
 14. The method of claim 10, wherein the mitogen activatedprotein kinase (MAPK) signal transduction pathway is activated in thecell.
 15. The method of claim 10, wherein the cell is a human cell. 16.The method of claim 10, wherein the cell is a Xenopus oocyte.
 17. Themethod of claim 10, wherein the cell is a cancer cell.
 18. The method ofclaim 17, wherein the cancer cell is from a sarcoma.
 19. The method ofclaim 10, wherein the cell is from a transformed cell line.
 20. Themethod of claim 19, wherein the cell line is transformed with Ras. 21.The method of claim 10, wherein the step of assaying comprises an MAPKK1or MAPKK2 mobility assay.
 22. The method of claim 10, wherein the stepof assaying comprises a Mos-induced activation of MAPK assay in aXenopus oocyte.
 23. The method of claim 10, wherein the MAPKK1 or MAPKK2is recombinant.
 24. The method of claim 23, wherein the recombinantMAPKK1 or recombinant MAPKK2 is linked to a detectable moiety.
 25. An invitro method for screening mimetics of lethal factor (LF) having mitogenactivated protein kinase kinase (MAPKK) protease activity, the methodcomprising the steps of: (i) providing a compound suspected of being anLF mimetic in an aqueous solution; and (ii) assaying for the level ofMAPKK protease activity.
 26. The method of claim 25, wherein the step ofassaying comprises a Mos-induced activation of MAPK assay in a Xenopusoocyte lysate.
 27. The method of claim 25, wherein the step of assayingcomprises an MAPKK1 or MAPKK2 mobility assay.
 28. The method of claim27, wherein the MAPKK1 or MAPKK2 is recombinant.
 29. The method of claim28, wherein the recombinant MAPKK1 or recombinant MAPKK2 is linked to adetectable moiety.
 30. The method of claim 25, wherein the step ofassaying comprises an myelin basic protein (MBP) phosphorylation assay.31. An in vivo method for screening mimetics of lethal factor (LF)having mitogen activated protein kinase kinase (MAPKK) proteaseactivity, the method comprising the steps of: (i) contacting a livingcell with a compound suspected of being an LF mimetic; and (ii) assayingfor the level of MAPKK protease activity.
 32. The method of claim 31,wherein the mitogen activated protein kinase (MAPK) signal transductionpathway is activated in the cell.
 33. The method of claim 31, whereinthe cell is a human cell.
 34. The method of claim 31, wherein the cellis a Xenopus oocyte.
 35. The method of claim 31, wherein the cell is acancer cell.
 36. The method of claim 35, wherein the cancer cell is froma sarcoma.
 37. The method of claim 31, wherein the cell is from atransformed cell line.
 38. The method of claim 37, wherein the cell lineis transformed with Ras.
 39. The method of claim 31, wherein the step ofassaying comprises an MAPKK1 or MAPKK2 mobility assay.
 40. The method ofclaim 31, wherein the step of assaying comprises a Mos-inducedactivation of MAPK assay in a Xenopus oocyte.
 41. The method of claim31, wherein the MAPKK1 or MAPKK2 is recombinant.
 42. A method forinhibiting proliferation of a cancer cell, the method comprising thestep of contacting the cell with LF, wherein the LF has MAPKK proteaseactivity.
 43. The method of claim 42, wherein the LF is recombinant. 44.The method of claim 42, wherein the step of contacting the cellcomprises transducing the cell with an expression vector encoding LF.45. The method of claim 42, wherein the step of contacting furthercomprises contacting a cell with LF in the presence of protectiveantigen (PA).
 46. The method of claim 45, wherein the PA is a fusionprotein targeted to the cancer cell.
 47. The method of claim 42, whereinthe mitogen activated protein kinase (MAPK) signal transduction pathwayis activated in the cancer cell.
 48. The method of claim 42, wherein thecell is a human cell.
 49. The method of claim 42, wherein the cancercell is from a sarcoma.
 50. The method of claim 42, wherein the cell isfrom a transformed cell line.
 51. The method of claim 50, wherein thecell line is transformed with Ras.
 52. In a computer system, a methodfor identifying a three-dimensional structure of LF proteins, the methodcomprising the steps of: (i) receiving input of at least 10 contiguousamino acids of the amino acid sequence of LF or at least 30 contiguousnucleotides of the nucleotide sequence of a gene encoding LF, andconservatively modified variants thereof; and (ii) generating athree-dimensional structure of the protein encoded by the amino acidsequence.
 53. The method of claim 52, wherein said amino acid sequenceis a primary structure and wherein said generating step includes thesteps of: (i) forming a secondary structure from said primary structureusing energy terms encoded by the primary structure; and (ii) forming atertiary structure from said secondary structure using energy termsencoded by said secondary structure.
 54. The method of claim 52, whereinsaid generating step includes the step of forming a quaternary structurefrom said tertiary structure using anisotropic terms encoded by thetertiary structure.
 55. The method of claim 53, wherein said generatingstep further includes the step of forming a quaternary structure fromsaid tertiary structure using anisotropic terms encoded by the tertiarystructure.
 56. The method of claim 52, further comprising the step ofidentifying regions of the three-dimensional structure of the proteinthat bind to ligands and using the regions to identify ligands that bindto the protein.
 57. In a computer system, a method for identifying athree-dimensional structure of MAPKK proteins, the method comprising thesteps of: (i) receiving input of at least 10 contiguous amino acids ofthe amino acid sequence of MAPKK or at least 30 contiguous nucleotidesof the nucleotide sequence of a gene encoding MAPKK, and conservativelymodified variants thereof; and (ii) generating a three-dimensionalstructure of the protein encoded by the amino acid sequence.
 58. Themethod of claim 57, wherein said amino acid sequence is a primarystructure and wherein said generating step includes the steps of: (i)forming a secondary structure from said primary structure using energyterms encoded by the primary structure; and (ii) forming a tertiarystructure from said secondary structure using energy terms encoded bysaid secondary structure.
 59. The method of claim 57, wherein saidgenerating step includes the step of forming a quaternary structure fromsaid tertiary structure using anisotropic terms encoded by the tertiarystructure.
 60. The method of claim 58, wherein said generating stepfurther includes the step of forming a quaternary structure from saidtertiary structure using anisotropic terms encoded by the tertiarystructure.
 61. The method of claim 57, further comprising the step ofidentifying regions of the three-dimensional structure of the proteinthat bind to ligands and using the regions to identify ligands that bindto the protein.